柔嫩艾美耳球虫烯醇化酶基因的克隆表达和抗原性分析

发布时间：2018-04-24 09:43

本文选题：柔嫩艾美耳球虫 + 烯醇化酶　；参考：《江苏农业学报》2017年03期

【摘要】：从纯化的柔嫩艾美耳球虫第二代裂殖子中提取总RNA,应用RT-PCR技术克隆获得了烯醇化酶基因(Gen Bank Accession No.GU169333)。烯醇化酶基因开放阅读框长1 338 bp,编码445个氨基酸。应用DNAstar软件对氨基酸序列的亲水区域和疏水区域的分布、抗原指数及T细胞基序进行分析,结果显示该蛋白质亲水性较强,抗原指数高,含较多T细胞表位。同时构建了烯醇化酶原核表达重组质粒p ET-28a(+)-ENO。用IPTG对重组质粒p ET-28a(+)-ENO进行诱导表达,SDS-PAGE结果显示表达的融合蛋白大小约为5.17×104,主要存在于裂解上清中。Western blotting结果显示该重组蛋白可被抗柔嫩艾美耳球虫的多克隆抗体识别,表明该蛋白质具有较好的免疫原性。
[Abstract]:Total RNAs were extracted from the purified second generation merozoites of Eimeria tenella. The enolase gene Gen Bank Accession no. GU169333 was cloned by RT-PCR technique. The open reading frame of enolase gene is 1 338 BP and encodes 445 amino acids. The distribution of hydrophilic and hydrophobic regions, antigen index and T cell motif of amino acid sequence were analyzed by DNAstar software. The results showed that the protein had strong hydrophilicity, high antigen index and more T cell epitopes. At the same time, the prokaryotic expression plasmid of enolase pET-28awas constructed. The recombinant plasmid pET-28a (pET-28a) was induced by IPTG and expressed by SDS-PAGE. The size of the fusion protein was about 5.17 脳 10 ~ 4, which was mainly found in the lytic supernatant. Western blotting showed that the recombinant protein could be recognized by polyclonal antibody against Eimeria tenella. The results showed that the protein had good immunogenicity.
【作者单位】：南京农业大学动物医学院;江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室;
【基金】：国家自然科学基金国际(地区)合作与交流项目(NSFCPSF,中巴)(31661143017);国家自然科学基金面上项目(31372428;31672545) 江苏高校优势学科建设工程资助项目(PAPD)
【分类号】：S852.723

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